2.1 Patient samples
After Institutional Review Board approval at the Catholic University of Korea, Seoul St. Mary’s Hospital (IRB No. KC18SESI0795), formalin-fixed, paraffin-embedded (FFPE) tissues were obtained from the Korean prostate Bank from patients with low- (n=22), intermediate- (n=41), and high-risk (n=36) PCa according to the National Comprehensive Cancer Network risk group [13] and from patients with BPH (n=28). Tumor tissue and the corresponding adjacent prostate tissue were collected separately from the PCa patients. All patients with PCa underwent radical prostatectomy, and their median follow-up duration was 38 months (range 6 to 55). We reviewed and collected the baseline demographics, clinicopathologic results, and follow-up outcomes.
2.2 Immunohistochemistry
Immunohistochemistry (IHC) was carried out to confirm the HMGB1 expression in the prostate tissues. The FFPE tissue sections were blocked and incubated with anti-HMGB1 antibody (Abcam, Cambridge, England) overnight at 4 °C. After washing, the samples were incubated with a secondary antibody conjugated to horseradish peroxidase (HRP) for two hours at room temperature and counterstained with hematoxylin. Digital images were obtained using an Olympus BX50 optical microscope (Olympus Optical Co. Ltd., Tokyo, Japan). The areas positively stained with HMGB1 (%) were measured by ParaView 1.15.1 (Sandia Corporation, Kitware Inc., Albuquerque, NM, USA).
2.3 Cell lines and culture
The RWPE1 cells were purchased from the American Type Culture Collection (Manassas, VA, USA), and cultured in complete Keratinocyte-Serum Free Media (K-SFM) (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 5 ng/ml epidermal growth factor and 50 µg/ml bovine pituitary extract. DU145, PC3, LNCaP, and HEK 293 cells were purchased from the Korean Cell Line Bank (Seoul, Korea). The DU145, PC3, and LNCaP cells were routinely maintained in RPMI 1640 medium (Gibco) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin. The HEK 293 cells were routinely maintained in DMEM medium (Gibco) containing 10% FBS and 1% penicillin/streptomycin. All cell lines were maintained at 37 °C in a humidified atmosphere of 5% CO2.
2.5 Transfection
Small-interfering RNAs (siRNAs) of human HMGB1 and control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Inc. CA, USA). DU145 and PC3 transfections were carried out using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) per the manufacturer’s protocol. Plasmid vectors (human HMGB1-Flag, HG10326-CF; tumor necrosis factor receptor-1/TNFR1-HA, HG10872-CY; TNFR3-HA, HG10581-CY; TNFR5-HA, HG10872-CY; and pCMV3 Negative Control, CV013) were purchased from Sino Biological (Sino Biological, Inc., Beijing, China). Transfection with plasmids was carried out using Lipofectamine 3000 (Invitrogen) per the manufacturer’s protocol.
2.6 Cell viability
The cells (DU145, 8 x103/well; PC3, 1 x104/well) were seeded in 96-well plates and then transfected with siRNAs in a CO2 incubator at 37 °C. At 0 h, 24 h, 48 h, and 72 h post-transfection, cell viability was measured using EZ-CYTOX (Daeil Lab Service Co. Ltd, Seoul, Korea) according to the manufacturer’s instructions.
2.7 Cell invasion assay
Cell invasion assays were performed using 24-well transwell chambers (8µm, Corning Inc., NY, USA) and the upper chambers were coated with 25µg/ml Matrigel (Corning). The cells were added to the pre-coated upper chambers at a density of 5 × 104 /well. Then, the lower chambers were filled with culture medium containing 20% FBS. After incubation in a CO2 incubator for 48 h at 37 °C, the cells on the lower surface of the upper chamber were fixed and stained with 0.1% crystal violet solution (Sigma-Aldrich, St. Louis, MO, USA). The membranes were observed under light microscopy and dissolved with 20% acetic acid. The solubilized color was measured at 570nm.
2.8 Flow cytometric analysis of cell cycle
Forty-eight hours after transfection, the cells were harvested and fixed with 70% cold ethanol at 4 °C for 1 h. After staining with 10 μg/ml propidium iodide and 10 mg/ml RNase at room temperature for 30 min in the dark, the percentage of cells in each cell cycle phase was determined using a FACS Canto II system (BD Biosciences, San Jose, CA, USA).
2.9 Western blot analysis
Forty-eight hours after transfection, the cells were harvested and lysed with RIPA buffer (Cell Signaling Technology, Danvers, MA, USA) containing a protease inhibitor. The protein concentrations were confirmed using a BCA Protein Assay Kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The proteins were separated by SDS-PAGE and then transferred onto nitrocellulose membranes. After transfer, the membranes were blocked and then incubated with primary antibodies against ERK (Cell Signaling Technology), phosphor-ERK (Cell Signaling Technology), Akt (Cell Signaling Technology), phospho-Akt (Cell Signaling Technology), Ikkβ (Abcam), cleaved-caspase-3 (Cell Signaling Technology), p65 (Abcam), and β-actin (1:2500, Santa Cruz) at 4°C overnight. After incubation, the membranes were washed and then incubated with a secondary antibody conjugated to HRP for 2 h at room temperature. The chemiluminescence method (Amersham, Arlington Heights, IL, USA) was used to develop the protein bands.
2.10 Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was prepared using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Total RNA (1µg) was reverse transcribed to cDNA using the Prime Script™ RT reagent Kit (Takara Bio Inc., Shiga, Japan) and the cDNA products were generated and amplified by RT-PCR. The PCR products were analyzed by electrophoresis on 1.5% agarose gels and visualized via a Gel Doc XR+ System (Bio-Rad, Hercules, CA, USA). The DNA sequences for the primer pairs used in RT-PCR are shown in Table 1.
2.11 Proteome profiler antibody array
The Proteome Profiler™ Human NF-κB Pathway Array (ARY029) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Proteome profiler antibody microarray analysis was performed according to the protocol provided. Briefly, 72 h after transfection, 500ug of each sample was incubated overnight at 4°C on the dot blot membrane of the human NF-κB pathway array. The membrane was washed with 1X washing buffer and incubated with a reconstituted detection antibody cocktail and horseradish peroxidase-conjugated streptavidin. The membranes were exposed to X-ray film and the mean intensity of each spot was quantified using Image-J software (Rasband W; National Institute of Health, Bethesda, MD, USA: http://rsbweb.nih.gov/ij/index.html).
2.12 Co-immunoprecipitation assay
For the co-immunoprecipitation (Co-IP) assay, plasmids were transfected into HEK293 cells using Lipofectamine 3000 reagent. One day after transfection, the cells were harvested and whole-cell extracts were incubated with Dynabead Protein G (Invitrogen) conjugated to an antibody against Flag overnight at 4°C. After washing the beads and eluting with 2X sample buffer, the immunoprecipitated sample was subjected to SDS-PAGE and Western blotting was conducted to detect HA (Abcam) and Flag (Abcam).
2.13 Statistical analysis
GraphPad Prism Software v5 (GraphPad Prism Software Inc., San Diego, CA, USA) and SPSS version 24.0 (SPSS Inc., Chicago, IL, USA) were used for the statistical analyses. The data are expressed as the mean (± standard deviation [SD]) for continuous variables and the number of patients (proportions) for dichotomous variables. The differences between the groups were examined by independent t-test, one-way analysis of variance (ANOVA) test followed by Tukey’s post-test, and the chi-squared test. Kaplan-Meier analysis with a log-rank test was performed to evaluate biochemical recurrence (BCR)-free survival. Value of p<0.05 was considered statistically significant.